The water-level fluctuating zone of the Three Gorges Reservoir (TGR) region contains several different land use types which have been altered or newly established since the commencement of the dam's operations. This study sought to examine the variation of soil microorganisms (the quantity of bacteria, actinomycetes and fungi), soil enzyme activity, soil nutrient content and the relationship among these factors under three land-use regimes in the Ruxi river basin area: grassland, abandoned farmland and farmland. 30 sampling plots from 11 grassland, 16 abandoned farmland and 3 farmland in Ruxi river basin (a typical region in the TGR area) were set up. All samples were collected in the same month and from the same elevation to control for seasonal and flooding influences on soil microbiology. Soil samples were collected from the 0-20 cm and 20-40 cm soil layers for each sampling plots. Results showed that there were significant differences among land uses in certain soil biochemical properties. Of the three types of land use patterns, abandoned farmland displayed the highest quantity of bacteria, while the amounts of fungi and actinomycetes were the greatest in grassland. The activity of hydrogen peroxidase, invertase, protease and urease were also higher in abandoned farmland than in the other two land patterns. However, phosphatase activity was significantly higher in farmland than in abandoned farmland and grassland. Total nitrogen (TN), available phosphorus (AP) and available potassium (AK) concentrations were significantly lower in grasslands compared to farmland, while the organic matter (OM) content was significantly higher there than in abandoned farmland and farmland. There were no significant differences among total potassium (TK), total phosphorus (TP) or available nitrogen (AN) content or protease activity under the three kinds of land use patterns. Soil depth only had a significant impact on the quantity of soil microbes, invertase activity, urease activity and OM content. Furthermore, the effect exerted by land use type in combination with soil depth was only significant in invertase activity. The pH value of the abandoned farmland was significantly higher than the other two patterns of land use. An analysis of correlation of the relationships among the soil biochemical characteristics indicated that the most significant association existed between the pH value and the soil biological characteristics, suggesting that pH is a decisive influence on the other biochemical parameters in this region. In addition, the quantity of fungi and actinomycetes in soil were significantly positively correlated with OM content underling the important role these species play in improving soil fertility in this area. Compared to abandoned farmland and farmland, grassland plays an important role in stabilizing the soil environment by retaining soil nutrients and improving soil fertility in the fluctuating zone of the TGR region. Therefore, it is suggested that more land should be converted from farmland to grassland.